Dynamic protein acetylation is essential for normal cell physiology and development. Indeed, defects in acetyltransferases are associated with a wide variety of human diseases. The MYST family of histone acetyltransferases are highly conserved, from yeast to man, and they serve as the catalytic subunits of large multi-protein complexes whose structural compositions are also conserved. MYST acetyltransferases are required for early mammalian embryonic development and aberrant rearrangements or regulation of MYST genes are associated with human cancers. We are investigating the molecular genetics of two signature members of the MYST family: the Esa1 enzyme of budding yeast, and the Myst2 enzyme of mouse and humans. ESA1 encodes the only essential histone acetyltransferase in budding yeast. It is the catalytic subunit of two multi-protein complexes, NuA4 and picNuA4. We recently made the surprising discovery that catalysis is not the essential function of Esa1, as previously believed. Our data argue, instead, that Esa1 is a molecular switch that uses the binding of Cofactor A to control currently uncharacterized essential functions. We will carry out experiments designed to understand what the essential function of Esa1 is doing, and how it is executed. We will study conditional mutants of ESA1 that specifically expose its essential function, and characterize phenotypes, gene expression patterns, and promoter chromatin structure under nonpermissive conditions. We will specifically challenge the molecular switch model using genetic suppressors and biochemical assays of protein conformation in recombinant picNuA4. The results of these experiments are poised to completely change the way we think about MYST family proteins. Myst2 (Hbo1) is a mammalian MYST family enzyme that serves as the catalytic subunit of multi-protein complexes that include members of the Ing and Jade tumor suppressor families. Based on our work and that of others, it is clear that Myst2 is required for DNA replication licensing, interacts with p53 to mediate stress signaling, and has roles in transcription. We recently discovered that homozygous Myst2 knockout mouse embryos arrest development at embryonic day E7.5, a stage at which rapid proliferation and extensive gene reprogramming are about to occur for gastrulation. Myst2 is the only MYST gene with this knockout phenotype and we propose that it is required for the burst of DNA replication, or gene reprogramming at this stage. We will characterize the gene expression pattern of wild type, heterozygous, and homozygous Myst2 knockout embryos to identify the genes and pathways dependent on Myst2 for development. We will characterize DNA replication licensing, S phase progression, DNA damage response, and protein occupancy at DNA replication origins to define the involvement of Myst2 is proliferation at this critical stage of development. Finally, we will use our knowledge of ESA1 to construct conditional knockout mice carrying mutant Myst2 alleles that will reveal if it also has essential non-enzymatic functions during development. These experiments will greatly expand our understanding of Myst2 function in regulating DNA replication and gene expression in early mammalian development. PUBLIC HEALTH RELEVANCE: MYST family protein complexes carry out functions that are essential for proper gene expression, DNA replication, DNA damage repair, and embryonic development. Failures in the function of MYST genes are associated with genome instability and many diseases including human cancers. Little is known about their range of functions and target pathways. The research proposed in this application is designed to uncover new principles in how these enzymes work, what they do, and how they do it.